Remedies for inflammatory bowel diseases

ABSTRACT

Antibodies against AILIM (also called ICOS and 8F4) were found to significantly suppress the onset of inflammatory bowel diseases (especially Crohn&#39;s disease and colitis (ulcerative colitis and such)), and exhibit a significant therapeutic effect against inflammatory bowel diseases.

TECHNICAL FIELD

The present invention relates to pharmaceutical compositions comprisinga substance having an activity to modulate the biological activity ofthe “activation inducible lymphocyte immunomodulatory molecule” (AILIM)(also known as “inducible costimulator” (ICOS)), especially the signaltransduction mediated by AILIM.

Specifically, the present invention relates to pharmaceuticalcompositions comprising a substance having an activity to regulate (forexample, inhibit) the proliferation of AILIM-expressing cells, celldeath (or apoptosis), or immune cytolysis, or to modulate (for example,inhibit) the production of a cytokine (for example, interferon-γ, orinterleukin-4) by AILIM-expressing cells.

More specifically, the present invention comprises substances having anactivity to U the signal transduction via AILIM, and particularlypreferably, substances that induce cell death, apoptosis, or depletionof AILIM-expressing cells. The present invention relates topharmaceutical compositions for suppressing, treating, or preventingdiseases accompanying abnormal immunity of the intestinal tract (forexample, inflammatory bowel diseases such as colitis (ulcerative colitisand such) and Crohn's disease, and alimentary allergies).

BACKGROUND ART

Mucous membranes of the gastrointestinal tract are constantly exposednot only to antigens derived from food and enterobacterial flora, butalso to various antigens existing in the outside world that are harmfulto the living body, such as pathogenic microorganisms. Therefore, thegastrointestinal mucous membranes exhibit a cytotoxic activity in orderto compete against such antigens harmful to the living body. Whilemaintaining the ability to secrete antibodies to neutralize toxins,these mucous membranes also have the unique immune mechanism ofsuppressing excessive immune reactions against antigens such as food andenterobacterial flora (this mechanism is called gastrointestinal mucosalimmunity or intestinal immunity). Specifically, normal mucosal immunityis established on the balance between positive immune responses againstpathogens and negative immune responses against non-pathogenic antigens.When this balance of immunological homeostatic maintenance collapses,inflammation, allergies, and infections occur, triggering the onset ofintestinal diseases generally termed Inflammatory Bowel Diseases (IBD)and alimentary allergies.

The most representative inflammatory bowel diseases are Crohn's disease(CD) and colitis (especially Ulcerative Colitis (UC)) . Both arediseases in which the pathogen cannot be specified and chronic andrecurrent attacks of abdominal pain and diarrhea occur, causingsignificant, long-term obstacles to the daily life of child and juvenilepatients. Furthermore, since colitis (especially ulcerative colitis) maybecome a causative for colon cancer, there is an urgent need forelucidating the pathogenesis of colitis and developing effectivetherapeutic methods.

Although various possibilities such as genetic and environmental factorshave been discussed concerning the mechanism of onset for inflammatorybowel diseases, recent studies indicate the strong possibility thatabnormal immunity of the intestinal tract (gastrointestinal mucosalimmunity) may be the cause. More specifically, an inflammation orallergy occurs in the intestinal mucous membranes due to the inductionof an excessive immune response that occurs for some reason againstantigens in the intestine that are normally non-pathogenic and have alow immunogenicity, resulting in the onset of an inflammatory boweldisease.

Furthermore, abnormal immunity against foreign pathogens, antigensderived from food, or autoantigens has been suggested to be deeplyinvolved in such inflammations and allergies of the intestine.Furthermore, recent studies have suggested the possibility that abnormalimmune responses towards certain indigenous bacteria manifest as chronicinflammatory reactions.

This mechanism of onset of inflammations and allergies of the intestinedue to abnormal immunity of the intestinal tract is supported byanalyses on the function and differentiation of T cells of patients aswell as the cytokine production pattern in lesions or serum.Furthermore, analysis of various recently developed animal models ofinflammatory intestinal diseases also reveal that abnormal mucosalimmunity causes chronic inflammation in the intestine (Gastroenterology,Vol. 109, p. 1344-1367, 1995).

For example, it is clear that T cells are deeply involved in the onsetof chronic enteritis since inflammation of the intestine developsspontaneously in T cell receptor (TCR) α-chain knockout mice(TCRα^(-/-))(Cell, Vol. 75, p. 275-282, 1993; J. Exp. Med., Vol. 183, p.847-856, 1996). In colitis of these TCRα^(-/-) mice, the production ofIFN-γ in the intestine is elevated, and in the initial stage ofinflammation, a rise in IL-1α and IL-1β levels is seen (LaboratoryInvestigation, Vol. 76, p. 385-397, 1997). Furthermore, TCBβ (β^(dim)) Tcells that have a specific Vβ subset and produce IL-4 can be seen in thedigestive tract and lymph nodes (Gastroenterology, Vol. 112, p.1876-1886, 1997). In this model, it is thought that a deficiency ofTCRαβ T cells causes an increase in the fraction of abnormal T cells,which then causes abnormal regulation of cytokine production, becoming amediator of inflammation.

In a model in which CD4⁺/CD45RB^(high) T cells are introduced to severecombined immunodeficient mice (SCID mice), severe enteritis accompanyinghyperplasia of a mucosal layer and infiltration of lymphocytes in theintestine are induced. However, this enteritis does not occur whenunfractionated CD4⁺ T cells are simultaneously introduced (J. Exp. Med.,Vol. 178, p. 237-244, 1993; Int. Immunol., Vol. 5, p. 1461-1471, 1993).CD4⁺ T cells of SCID mice that have developed enteritis produce IFN-γ.On the other hand, since enteritis is suppressed by the administrationof antibodies against INF-γ, Th1 type T cells are considered to causethe inflammation (Immunity, Vol. 1, p. 553-562, 1994).

Based on these facts, there seems to be no doubt that CD4⁺ T cells ofthe intestine and excessive activation thereof are important factors ininflammatory bowel diseases.

Furthermore, regression of enteritis with the decrease in CD4⁺ T cellsin patients affected by both an inflammatory bowel disease and HIV, alsosupports the deep involvement of abnormal CD4⁺ T cells in inflammatorybowel diseases (J. Clin. Gastroenterology, Vol. 23, p. 24-28, 1996).Based on this finding, there have been attempts to treat inflammatorybowel diseases using an anti-CD4 antibody, and it has been reported thatinflammatory lesions are suppressed by the administration of ananti-CD4antibody(Gut, Vol. 40, p. 320-327, 1997).

On the other hand, such abnormal functional regulation of T cells meansthat the balance of regulatory cytokine production has collapsed.

In fact, enteritis is also reported to develop spontaneously inIL-2knockout mice and IL-10 knockout mice (Cell, Vol. 75, p. 235-261,1993; Cell, Vol. 75, p. 263-274, 1993). Furthermore, in these models,excess production of IFN-γ is also observed, supporting the fact that anexcessive Th1 type T cell reaction has occurred. Overproduction of IFN-γin these models is consistent with the observation of increasedexpression of IFN-γ in lesions seen in Crohn's disease. Enteritis can betreated in IL-10 deficient mice by administering IL-10. It has beenreported that enteritis can be suppressed by this method in SCID mice towhich CD4⁺/CD45RB^(high) T cells have been introduced (Immunity, Vol. 1,p. 553-562, 1994).

As mentioned above, the analysis of the mechanism of onset ofinflammatory bowel diseases has progressed from the aspect of abnormalgastrointestinal mucosal immunity, suggesting the possibility oftreating inflammatory bowel diseases by suppressing increased activationof CD4⁺ T cells and overproduced cytokines. However, the realpathogenesis of inflammatory bowel diseases has not yet been revealed,and furthermore, an effective therapeutic method has not been provided.

The activation of T cells (acquisition of antigen specificity) isinitiated when T cells recognize antigens presented byantigen-presenting cells (APCs) such as macrophages, B cells, ordendritic cells. APCs process the incorporated antigens, and theprocessed antigens are bound to the major histocompatibility antigencomplex (MHC) and presented. T cells receive the first signal for cellactivation (acquisition of specificity) as a result of the recognitionof the processed antigen presented by APCs through a complex formedbetween the T cell receptor (TCR) on the T cell membrane surface and theantigen (TCR/CD3 complex).

For sufficient activation of T cells, a second signal called thecostimulatory signal is necessary in addition to the first signal. Tcells are activated antigen-specifically by receiving this costimulatorysignal after receiving the first signal.

For this second signal transduction, the interaction (more specifically,the intercellular adhesion mediated by bonds formed between thefollowing molecules) among CD28 (also known as Tp44, T44, or 9.3antigen), which is a cell surface molecule expressed mainly in T cellsand thymus cells, CD80 (also known as B7-1, B7, BB1, or B7/BB1), whichis a cell surface molecule expressed by antigen-presenting cells(macrophages, monocytes, dendritic cells, etc.), and CD86 (also known asB7-2 or B70), which is also a cell surface molecule onantigen-presenting cells, is extremely important.

Furthermore, it has been experimentally revealed that the interaction(specifically, the intercellular adhesion mediated by bonds formedbetween the following molecules) among Cytolytic T Lymphocyte-associatedAntigen 4 (CTLA-4) whose expression is enhanced depending on the secondsignal, CD80 (B7-1), and CD86 (B7-2) also has an important role in theregulation of T cell activation by this second signal. Morespecifically, the regulation of T cell activation by this second signaltransduction has been revealed to include at least the interactionbetween CD28 and CD80/CD86, enhancement of the expression of CTLA-4considered to be dependent on this interaction, and the interactionbetween CTLA-4 and CD80/CD86.

In addition, recently, similarly to CTLA4 and CD28 described above, amolecule called activation inducible lymphocyte immunomodulatorymolecule (AILIM; human, mouse, and rat; Int. Immunol., 12(1), p. 51-55,2000; also called Inducible co-stimulator (ICOS; human; Nature,397(6716), p. 263-266, 1999); J. Immunol., 166(1), p. 1, 2001; J.Immunol., 165(9), p. 5035, 2000; Biochem. Biophys. Res. Commun., 276(1),p. 335, 2000; Immunity, 13(1), p. 95, 2000; J. Exp. Med., 192(1), p. 53,2000; Eur. J. Immunol., 30(4), p. 1040, 2000) was identified as thethird costimulatory transmission molecule that transduces a secondsignal (costimulatory signal) necessary for the activation oflymphocytes such as T cells, and coupled with the signal, regulates thefunction of activated lymphocytes such as activated T cells.

Furthermore, a novel molecule called B7h, B7RP-1, GL50, or LICOS whichis considered to be a ligand interacting with the costimulatorytransmission molecule AILIM has been identified (Nature. Vol. 402, No.6763, pp. 827-832, 1999; Nature Medicine, Vol. 5, No. 12, pp. 1365-1369,1999; J. Immunology, Vol. 164, pp. 1653-1657, 2000; Curr. Biol., Vol.10, No. 6, pp. 333-336, 2000).

Exhaustive studies are in progress on the biological functions of thesetwo novel molecules, the functional control of lymphocytes such as Tcells through the third costimulatory signal transduction by themolecules.

On the other hand, there has not been even suggestions on therelationship between AILIM (ICOS), which is the third costimulatorytransduction molecule considered essential for the activation of T cellssuch as CD4⁺ T cells, and the onset of the above-mentioned abnormalimmunity of the intestinal mucous membrane and inflammatory boweldiseases (Crohn's disease and colitis (ulcerative colitis and such)) .Neither has there been any suggestion on attempts to treat inflammatorybowel diseases by regulating the function of this AILIM molecule.

DISCLOSURE OF THE INVENTION

Specifically, an objective of the present invention is to providemethods and pharmaceutical agents for suppressing, treating, orpreventing diseases accompanying abnormal immunity (abnormal T cellactivation, increase of abnormal CD4⁺ cells) of the intestinal tractsuch as inflammatory bowel diseases (Crohn' s disease and colitis(ulcerative colitis and such)) by modulating, via medicinal andpharmaceutical methods (for example, pharmaceutical agents such as lowmolecular weight compounds and antibodies), the biological function ofthe novel molecule AILIM, which is considered to transduce the secondsignal essential for the activation of lymphocytes such as T cells(costimulatory signal) and regulate the function of activatedlymphocytes such as activated T cells.

A further objective is to use such pharmaceutical agents that modulatethe biological function of AILIM (for example, pharmaceutical agentssuch as low molecular weight compounds and antibodies) to providemethods for enhancing the therapeutic effect of existing pharmaceuticalagents widely used for treating inflammatory bowel diseases(adrenocortical hormones, salazosulfapyridine, etc.).

Extensive studies on methods for suppressing the biological function ofmammalian AILIM (ICOS), and alimentary allergies and inflammatory boweldiseases in which abnormal immunity of the intestinal tract may bedeeply involved (especially Crohn's disease and colitis (ulcerativecolitis and such)), led the present inventors to discover thatpharmaceutical agents that regulate the function of AILIM significantlysuppress inflammatory bowel diseases (especially Crohn's disease andcolitis (ulcerative colitis and such)). Thus, the present invention wasachieved.

A pharmaceutical composition of the present invention is useful as apharmaceutical for modulating various reactions in vivo in which thetransduction of a costimulatory signal to AILIM-expressing cellsmediated by AILIM is involved (for example, proliferation ofAILIM-expressing cells, production of cytokine(s) by AILIM-expressingcells, immune cytolysis or cell death, apoptosis, or depletion ofAILIM-expressing cells, and the activity to induce antibody-dependentcellular cytotoxicity against AILIM-expressing cells), and/or as apharmaceutical for preventing the onset and/or progression of variousdiseases in which the signal transduction mediated by AILIM is involved,and for the treatment or prophylaxis of the diseases.

Specifically, a pharmaceutical composition of the present invention canmodulate (suppress or promote) the proliferation of AILIM-expressingcells, apoptosis, cell death, or depletion, or immune cytolysis, or canmodulate (inhibit or promote) the production of cytokines (for example,interferon γ, or interleukin 4) by AILIM-expressing cells, and canprevent various disease conditions triggered by various physiologicalphenomena in which the signal transduction mediated by AILIM isinvolved, and enables the treatment or prevention of various diseases.

Such an especially preferred embodiment of the pharmaceuticalcompositions of this invention are pharmaceutical compositionscomprising a substance that induces cell death, apoptosis, or depletionof AILIM expressing cells.

Using the pharmaceutical compositions of this invention, diseases thatmay be caused by an abnormal immunity of the intestinal tract, morespecifically, inflammatory bowel diseases (especially Crohn's diseaseand colitis (ulcerative colitis and such)) and alimentary allergies canbe suppressed, prevented, and/or treated.

Furthermore, the pharmaceutical compositions of this invention canenhance the therapeutic effect on inflammatory bowel diseases when usedin combination with an existing pharmaceutical agent prescribed to treatsuch inflammatory bowel diseases.

More specifically, the present invention is as described in thefollowing (1) to (10).

(1) A pharmaceutical composition for suppressing, treating, orpreventing a disease that accompanies abnormal immunity of theintestinal tract, wherein the pharmaceutical composition comprises asubstance having an activity to modulate signal transduction via AILIMand a pharmaceutically acceptable carrier.

(2) The pharmaceutical composition of (1) wherein said substance has anactivity to induce cell death of an AILIM-expressing cell.

(3) The pharmaceutical composition of (1) or (2), wherein said diseaseis an inflammatory bowel disease.

(4) The pharmaceutical composition of (3), wherein said inflammatorybowel disease is colitis.

(5) The pharmaceutical composition of (3), wherein said inflammatorybowel disease is Crohn's disease.

(6) The pharmaceutical composition of (1) or (2), wherein said diseaseis an alimentary allergy.

(7) The pharmaceutical composition of any one of (1) to (6), whereinsaid substance is a proteinaceous substance.

(8) The pharmaceutical composition of (7) wherein said proteinaceoussubstance is selected from group consisting of:

a) an antibody that binds to AILIM, or a part of said antibody;

b) a polypeptide comprising the whole extracellular region of AILIM, ora part thereof;

c) a fusion polypeptide comprising the whole or a portion ofextracellular region of AILIM, and the whole or a portion of constantregion of the immunoglobulin heavy chain; and,

d) a polypeptide that binds to AILIM. (9) The pharmaceutical compositionof any one of (1) to (6), wherein said substance is a non-proteinaceoussubstance. (10) The pharmaceutical composition of (9) wherein saidnon-proteinaceous substance is DNA, RNA, or a chemically synthesizedcompound.

The present inventions are described in detail herein below by definingthe terms and the methods for producing the substances used in thisinvention.

Herein, the term “mammal” means a human, cow, goat, rabbit, mouse, rat,hamster, and guinea pig; preferred is a human, cow, rat, mouse, orhamster, and particularly preferred is a human.

“AILIM” of this invention is an abbreviation for “Activation InducibleLymphocyte Immunomodulatory Molecule” and denotes a cell surfacemolecule of a mammal having the structure and function described inprevious reports (J. Immunol., 166(1), p. 1, 2001; J. Immunol., 165(9),p. 5035, 2000; Biochem. Biophys. Res. Commun., 276(1), p. 335, 2000;Immunity, 13(1), p. 95,2000; J. Exp. Med., 192(1), p. 53, 2000; Eur. J.Immunol., 30(4), p. 1040, 2000; Int. Immunol., 12(1), p. 51, 2000;Nature, 397(6716), p. 263, 1999; GenBankAccession Number: BAA82129(human); BAA82128 (rat); BAA82127 (mutant rat); BAA82126 (mouse)).

Especially preferably, the term denotes AILIM derived from a human (forexample, International Immunology, Vol. 12, No. 1, p. 51-55, 2000;GenBank Accession Number: BAA82129).

This AILIM is also called ICOS (Nature, Vol. 397, No. 6716, p. 263-266,1999) or JTT-1 antigen/JTT-2 antigen (Unexamined Published JapanesePatent Application No. (JP-A) Hei 11-29599, International PatentApplication No. WO98/38216), and these molecules mutually refer to thesame molecule.

In addition, “AILIM” in this invention includes the amino acid sequencesof AILIM from each mammal described in previously reported literature,and especially preferably, a polypeptide having substantially the sameamino acid sequence as that of human AILIM. Furthermore, human AILIMmutants similar to the previously identified AILIM mutant derived fromrat (GenBank Accession Number: BAA82127) are also included in the“AILIM” of this invention.

Herein, the expression “having substantially the same amino acidsequence” means that “AILIM” of the present invention includespolypeptides having an amino acid sequences in which multiple aminoacids, preferably 1 to 10 amino acids, particularly preferably 1 to 5amino acids, have been substituted, deleted, and/or modified, andpolypeptides having an amino acid sequences in which multiple aminoacids, preferably 1 to 10 amino acids, particularly preferably 1 to 5amino acids, have been added, as long as the polypeptides havesubstantially the same biological properties as the polypeptidecomprising the amino acid sequence shown in previous reports.

Such substitutions, deletions, or insertions of amino acids can beachieved according to the usual method (Experimental Medicine:SUPPLEMENT, “Handbook of Genetic Engineering” (1992), etc.).

Examples are synthetic oligonucleotide site-directed mutagenesis (gappedduplex method), point mutagenesis by which a point mutation isintroduced at random by treatment with nitrite or sulfite, the method bywhich a deletion mutant is prepared with Bal31 enzyme and so on,cassette mutagenesis, linker scanning method, misincorporation method,mismatch primer method, DNA segment synthesis method, etc.

Synthetic oligonucleotide site-directed mutagenesis (gapped duplexmethod) can be performed, for example, as follows. The region one wishesto mutagenize is cloned into a M13 phage vector having an amber mutationto prepare a single-stranded phage DNA. After RF I DNA of M13 vectorhaving no amber mutation is linearized by restriction enzyme treatment,the DNA is mixed with the single-stranded phage DNA mentioned above,denatured, and annealed thereby forming a “gapped duplex DNA.” Asynthetic oligonucleotide into which mutations are introduced ishybridized with the gapped duplex DNA and a closed-circulardouble-stranded DNA is prepared by reacting with DNA polymerase and DNAligase. E. coli mutS cells, deficient in mismatch repair activity, aretransfected with this DNA. E. coli cells having no suppressor activityare infected with the grown phages, and only phages having no ambermutations are screened.

The method by which a point mutation is introduced with nitriteutilizes, for example, the principle as mentioned below. If DNA istreated with nitrite, nucleotides are deaminated to change adenine intohypoxanthine, cytosine into uracil, and guanine into xanthine. Ifdeaminated DNA is introduced into cells, “A:T” and “G:C” are replacedwith “G:C” and “A:T”, respectively, because hypoxanthine, uracil, andxanthine base pair with cytosine, adenine, and thymine, respectively, inDNA replication. Actually, single-stranded DNA fragments treated withnitrite are hybridized with “gapped duplex DNA”, and thereafter, mutantstrains are separated by manipulating in the same way as syntheticoligonucleotide site-directed mutagenesis (gapped duplex method).

The term “cytokine” as in “production of a cytokine by AILIM-expressingcells” in the present invention means an arbitrary cytokine produced byAILIM-expressing cells (especially, T cells).

Examples of T cells are T cells of the Th1 type or Th2 type, and acytokine of the present invention specifically means a cytokine producedby T cells of the Th1 type and/or an arbitrary cytokine produced by Tcells of the Th2 type.

Cytokines produced by T cells of the Th1type include IFN-γ, IL-2, TNF,IL-3, and cytokines produced by T cells of Th2 type include IL-3, IL-4,IL-5, IL-10, and TNF (Cell, Vol. 30, No. 9, pp. 343-346, 1998).

The expression “substance”, “substance having an activity to modulatethe signal transduction mediated by AILIM”, “substance having anactivity to inhibit the proliferation of AILIM-expressing cells, or toinhibit the production of a cytokine by AILIM-expressing cells”, or“substance having an activity to induce cell death of AILIM-expressingcells” as used in the present invention means a naturally-occurringsubstance or an artificially-prepared arbitrary substance.

Particularly preferred embodiment of the “substance” according to thisinvention is the substance having an activity to induce cell death,apoptosis, or depletion of AILIM-expressing cells.

Herein, the expression “signal transduction mediated by AILIM” meanssignal transduction through AILIM, leading to a change of any phenotypein the AILIM-expressing cells described above or in the followingExamples (a change in cell proliferation, activation of cells,inactivation of cells, apoptosis, and/or the ability to produce anarbitrary cytokine from AILIM-expressing cells). “The substance” can bemainly classified into a “proteinaceous substance” and a“non-proteinaceous substance”.

Examples of “proteinaceous substances” are the following polypeptides,antibodies (polyclonal antibodies, monoclonal antibodies, or portions ofmonoclonal antibodies).

When the substance is an antibody, it is preferably a monoclonalantibody. When the substance is a monoclonal antibody, it includes notonly non-human mammal-derived monoclonal antibodies, but also thefollowing recombinant chimeric monoclonal antibodies, recombinanthumanized monoclonal antibodies, and human monoclonal antibodies.

When the substance is a polypeptide, it includes the followingpolypeptides, polypeptide (oligopeptide) fragments, fusion polypeptides,and chemically modified polypeptides. Examples of oligopeptides arepeptides comprising 5 to 30 amino acids, preferably 5 to 20 amino acids.A chemical modification can be designed depending on various purposes,for example, to increase half-life in blood in the case of administeringin vivo, or to increase tolerance against degradation, or increaseabsorption in the digestive tract in oral administrations.

Examples of polypeptides are as follows:

(1) A polypeptide containing the whole or a portion of extracellularregion of AILIM;(2) A fusion polypeptide comprising the whole or a portion ofextracellular region of AILIM, and the whole or a portion of constantregion of the immunoglobulin heavy chain; or(3) A polypeptide that binds to AILIM.

Examples of “non-proteinaceous substances” are DNA, RNA, and chemicallysynthesized compounds.

Here, “DNA” means “DNA comprising a partial nucleotide sequence of anantisense DNA designed based on the nucleotide sequence of the DNA(including cDNA and genomic DNA) encoding the above AILIM (preferablyhuman AILIM), or a chemically modified DNA thereof” useful as anantisense DNA pharmaceutical. Specifically, the antisense DNA caninhibit the transcription of DNA encoding AILIM into mRNA, or thetranslation of the mRNA into a protein by hybridizing to the DNA or RNAencoding AILIM.

The expression “partial nucleotide sequence” as referred to hereinrefers to a partial nucleotide sequence comprising an arbitrary numberof nucleotides in an arbitrary region. A partial nucleotide sequenceincludes 5 to 100 consecutive nucleotides, preferably 5 to 70consecutive nucleotides, more preferably 5 to 50 consecutivenucleotides, and even more preferably, 5 to 30 consecutive nucleotides.

When the DNA is used as an antisense DNA pharmaceutical, the DNAsequence can be chemically modified in part in order to extend thehalf-life (stability) in blood when the DNA is administered to patients,to increase the intracytoplasmic-membrane permeability of the DNA, or toincrease the degradation resistance or the absorption of orallyadministered DNA in the digestive organs. Chemical modificationsinclude, for example, the modification of a phosphate bond, a ribose, anucleotide, the sugar moiety, and the 3′ end and/or the 5′ end in thestructure of an oligonucleotide DNA.

Modifications of phosphate bonds include, for example, the conversion ofone or more bonds to phosphodiester bonds (D-oligo), phosphorothioatebonds, phosphorodithioate bonds (S-oligo), methyl phosphonate (MP-oligo)bonds, phosphoroamidate bonds, non-phosphate bonds or methylphosphonothioate bonds, or combinations thereof. Modification of aribose includes, for example, the conversion to 2′ -fluororibose or 2′-O-methylribose. Modification of a nucleotide includes, for example, theconversion to 5-propynyluracil or 2-aminoadenine.

Here, the term “RNA” means “RNA comprising a partial nucleotide sequenceof an antisense RNA designed based on the nucleotide sequence of the RNAencoding the above AILIM (preferably human AILIM), or a chemicallymodified RNA thereof” useful as an antisense RNA pharmaceutical. Theantisense RNA can inhibit the transcription of the DNA encoding AILIMinto mRNA, or the translation of the mRNA into a protein by hybridizingto the DNA or RNA encoding AILIM.

The expression “partial nucleotide sequence” as employed herein, refersto a partial nucleotide sequence comprising an arbitrary number ofnucleotides in an arbitrary region. A partial nucleotide sequenceincludes 5 to 100 consecutive nucleotides, preferably 5 to 70consecutive nucleotides, more preferably 5 to 50 consecutivenucleotides, and even more preferably 5 to 30 consecutive nucleotides.

The antisense RNA sequence can be chemically modified in part in orderto extend the half-life (stability) in blood when the RNA isadministered to patients, to increase the intracytoplasmic-membranepermeability of the RNA, or to increase the degradation resistance orthe absorption of orally administered RNA in digestive organs. Chemicalmodifications include modifications such as those that apply to theabove antisense DNA.

Examples of “a chemically synthesized compound” are an arbitrarycompound excluding the above DNA, RNA and proteinaceous substances,having a molecular weight of about 100 to about 1000, or less,preferably a compound having a molecular weight of about 100 to about800, and more preferably a molecular weight of about 100 to about 600.

The term “polypeptide” included in the definition of the above“substance” means a portion (a fragment) of a polypeptide chainconstituting AILIM (preferably human AILIM), preferably the whole or aportion of an extracellular region of the polypeptide constituting AILIM(1 to 5 amino acids may be optionally added into the N-terminus and/orC-terminus of the region).

AILIM according to the present invention is a transmembrane moleculepenetrating the cell membrane, comprising 1 or 2 polypeptide chains.

Herein, a “transmembrane protein” means a protein that is connected tothe cell membrane through a hydrophobic peptide region that penetratesthe lipid bilayer of the membrane once or several times, and whosestructure is, as a whole, composed of three main regions, that is, anextracellular region, a transmembrane region, and a cytoplasmic region,as seen in many receptors or cell surface molecules. Such atransmembrane protein constitutes each receptor or cell surface moleculeas a monomer, or as a homodimer, heterodimer or oligomer coupled withone or several chains having the same or different amino acidsequence(s).

Here, an “extracellular region” means the whole or a portion of apartial structure (partial region) of the entire structure of theabove-mentioned transmembrane protein where the partial structure existsoutside of the membrane. In other words, it means the whole or a portionof the region of the transmembrane protein excluding the regionincorporated into the membrane (transmembrane region) and the regionexisting in the cytoplasm following the transmembrane region(cytoplasmic region). “A fusion polypeptide” included in the above“proteinaceous substance” means a fusion polypeptide comprising thewhole or a portion of the extracellular region of a polypeptideconstituting AILIM (preferably human AILIM), and “the whole or a portionof the constant region of immunoglobulin heavy chain (Ig, preferablyhuman Ig)”. Preferably, the fusion polypeptide is a fusion polypeptidehaving the extracellular region of AILIM and a portion of the constantregion of human IgG heavy chain, and particularly preferably, a fusionpolypeptide of the extracellular region of AILIM and a region (Fc) ofhuman IgG heavy chain comprising a hinge region, C_(H)2 domain andC_(H)3 domain. As an IgG, IgG1 is preferable, and as AILIM, human,mouse, or rat AILIM is preferable (preferably human).

The expression “the whole or a portion of the constant region ofimmunoglobulin (Ig) heavy chain” as used herein means the constantregion or the Fc region of human-derived immunoglobulin heavy chain (Hchain), or a portion thereof. The immunoglobulin can be anyimmunoglobulin belonging to any class and any subclass. Specifically,the immunoglobulin includes IgGs (IgG1, IgG2, IgG3, and IgG4), IgM, IgAs(IgA1 and IgA2), IgD, and IgE. Preferably, the immunoglobulin is IgG(IgG1, IgG2, IgG3, or IgG4), or IgM. Examples of particularly preferableimmunoglobulins of the present invention are those belonging tohuman-derived IgGs (IgG1, IgG2, IgG3, or IgG4).

Immunoglobulin has a Y-shaped structural unit in which four chainscomposed of two homologous light chains (L chains) and two homologousheavy chains (H chains) are connected through disulfide bonds (S-Sbonds). The light chain is composed of the light chain variable region(V_(L)) and the light chain constant region (C_(L)). The heavy chain iscomposed of the heavy chain variable region (V_(H)) and the heavy chainconstant region (C_(H)).

The heavy chain constant region is composed of some domains having aminoacid sequences unique to each class (IgG, IgM, IgA, IgD, and IgE) andeach subclass (IgG1, IgG2, IgG3, and IgG4, IgA1, and IgA2).

The heavy chain of IgGs (IgG1, IgG2, IgG3, and IgG4) is composed ofV_(H), C_(H)1 domain, hinge region, C_(H)2 domain, and C_(H)3 domain inthis order from the N-terminus.

Similarly, the heavy chain of IgG1 is composed of V_(H), Cγ₁1 domain,hinge region, Cγ₁2 domain, and Cγ₁3 domain in this order from the Nterminus. The heavy chain of IgG2 is composed of V_(H), Cγ_(b 2)1domain, hinge region, Cγ₂2 domain, and Cγ₂3 domain in this order fromthe N-terminus. The heavy chain of IgG3 is composed of V_(H), Cγ₃1domain, hinge region, Cγ₃2 domain, and Cγ₃3 domain in this order fromthe N terminus. The heavy chain of IgG4 is composed of V_(H), Cγ₄1domain, hinge region, Cγ₄2 domain, and Cγ₄3 domain in this order fromthe N-terminus.

The heavy chain of IgA is composed of V_(H), Cα1 domain, hinge region,Cα2 domain, and Cα3 domain in this order from the N-terminus.

Similarly, the heavy chain of IgA1 is composed of V_(H), Cα₁1 domain,hinge region, Cα₁2 domain, and Cα₁3 domain in this order from theN-terminus. The heavy chain of IgA2 is composed of V_(H), Cα₂1 domain,hinge region, Cα₂2 domain, and Cα₂3 domain in this order from theN-terminus.

The heavy chain of IgD is composed of V_(H), Cδ1 domain, hinge region,Cδ2 domain, and Cδ3 domain in this order from the N-terminus.

The heavy chain of IgM is composed of V_(H), Cμ1 domain, Cμ2 domain, Cμ3domain, and Cμ4 domain in this order from the N-terminus and has nohinge region as seen in IgG, IgA, and IgD.

The heavy chain of IgE is composed of V_(H), Cε1 domain, Cε2 domain, Cε3domain, and Cε4 domain in this order from the N-terminus and have nohinge region as seen in IgG, IgA, and IgD.

If, for example, IgG is treated with papain, it is cleaved at a slightlyN-terminal side beyond the disulfide bonds existing in the hinge regionwhere the disulfide bonds connect the two heavy chains to generate twohomologous Fabs, in which a heavy chain fragment composed of V_(H) andC_(H)1 is connected to one light chain through a disulfide bond; and oneFc, in which two homologous heavy chain fragments composed of the hingeregion, C_(H)2 domain, and C_(H)3 domain are connected through disulfidebonds (See “Immunology Illustrated”, original 2nd ed., Nankodo, pp.65-75 (1992); and “Focus of Newest Medical Science ‘RecognitionMechanism of Immune System’”, Nankodo, pp. 4-7 (1991); and so on).

Namely, “a portion of the constant region of immunoglobulin heavy chain”mentioned above means a portion of the constant region of animmunoglobulin heavy chain having the structural characteristics asmentioned above, and preferably, is a constant region without the C1domain, or the Fc region. Specifically, an example thereof is a regioncomposed of the hinge region, C2 domain, and C3 domain from each of IgG,IgA, and IgD, or is a region composed of C2 domain, C3 domain, and C4domain from each of IgM and IgE. A particularly preferable examplethereof is the Fc region of human-derived IgG1.

The fusion polypeptide mentioned above has the advantage of beingextremely easy to purify by using affinity column chromatography usingthe property of protein A, which binds specifically to theimmunoglobulin fragment, because the fusion polypeptide of the presentinvention has a portion of a constant region (for example Fc) of animmunoglobulin such as IgG as mentioned above as a fusion partner.Moreover, since various antibodies against the Fc of variousimmunoglobulins are available, an immunoassay for the fusionpolypeptides can be easily performed with antibodies against the Fc.

“A polypeptide that binds to AILIM” is encompassed in “a polypeptide”included in the definition of the above “substance”.

A specific example of “a polypeptide that binds to AILIM” is the wholeor a portion of a polypeptide constituting known molecule called B7h,B7RP-1, GL50, or LICOS which is a ligand interacting with AILIM (Nature,Vol. 402, No. 6763, pp. 827-832, 1999; Nature Medicine, Vol. 5, No. 12,pp. 1365-1369, 1999; J. Immunology, Vol. 164, pp. 1653-1657, 2000; Curr.Biol., Vol. 10, No 6, pp. 333-336, 2000).

Preferably, the polypeptide is a polypeptide comprising the whole or aportion of an extracellular region of the above ligand (B7h, B7RP-1,GL50, LICOS), or a fusion polypeptide comprising the polypeptide, andthe whole or a portion of the constant region of immunoglobulin heavychain (preferably human immunoglobulin). Here, the expressions“extracellular region” and “constant region of immunoglobulin heavychain” have the same meanings as mentioned above.

The polypeptides, portions of the polypeptide (fragment), and fusionpolypeptides mentioned above can be produced not only by recombinant DNAtechnology as mentioned below, but also by a method well known in theart such as a chemical synthetic method or a cell culture method, or amodified method thereof.

The “antibody” of the present invention can be a polyclonal antibody(antiserum) or a monoclonal antibody against mammalian AILIM(particularly preferably human AILIM) defined above, and preferably amonoclonal antibody.

Specifically, the antibody is an antibody having an activity to inhibitproliferation of AILIM-expressing cells by binding to AILIM, or toinhibit production of interferon-γ or interleukin-4 by AILIM-expressingcells through binding to AILIM.

The antibodies of the present invention can be natural antibodiesobtained by immunizing mammals such as mice, rats, hamsters, guineapigs, and rabbits with an antigen such as cells (natural cells, celllines, tumor cells, etc.) expressing AILIM of the present invention,transformants prepared using recombinant DNA technology so as tooverexpress AILIM on the surface thereof, polypeptides constitutingAILIM, or the above-mentioned fusion polypeptides comprising the AILIMpolypeptide or the extracellular region of AILIM. The antibodies of thepresent invention also include chimeric antibodies and humanizedantibodies (CDR-grafted antibodies) that can be produced by recombinantDNA technology, and human antibodies that can be produced using humanantibody-producing transgenic animals.

Monoclonal antibodies include those having any one isotype of IgG, IgM,IgA, IgD, or IgE. IgG or IgM is preferable.

A polyclonal antibody (antisera) or monoclonal antibody can be producedby known methods. Namely, a mammal, preferably, a mouse, rat, hamster,guinea pig, rabbit, cat, dog, pig, goat, horse, or cow, or morepreferably, a mouse, rat, hamster, guinea pig, or rabbit is immunized,for example, with an antigen mentioned above with Freund's adjuvant, ifnecessary.

A polyclonal antibody can be obtained from the serum obtained from theanimal so immunized. In addition, monoclonal antibodies are produced asfollows. Hybridomas are prepared from the antibody-producing cellsobtained from the animal so immunized and myeloma cells that are notcapable of producing autoantibodies. The hybridomas are cloned, andclones producing the monoclonal antibodies showing a specific affinityto the antigen used for immunizing the mammal are screened.

Specifically, a monoclonal antibody can be produced as follows.Immunizations are performed by injecting or implanting once or severaltimes an antigen mentioned above as an immunogen, if necessary, withFreund's adjuvant, subcutaneously, intramuscularly, intravenously,through the footpad, or intraperitoneally into a non-human mammal,specifically a mouse, rat, hamster, guinea pig, or rabbit, preferably amouse, rat, or hamster (including a transgenic animal generated so as toproduce antibodies derived from another animal such as a transgenicmouse producing human antibody mentioned below) . Usually, immunizationsare performed once to four times every one to fourteen days after thefirst immunization. Antibody-producing cells are obtained from themammal so immunized in about one to five days after the lastimmunization. The frequency and interval of immunizations can beappropriately arranged depending on, for example, the property of theimmunogen used.

Hybridomas that secrete a monoclonal antibody can be prepared by themethod of Köhler and Milstein (Nature, Vol. 256, pp. 495-497 (1975)), orby a modified method thereof. Namely, hybridomas are prepared by fusingantibody-producing cells contained in a spleen, lymph node, bone marrow,or tonsil obtained from a non-human mammal immunized as mentioned above,preferably a spleen, with myelomas without an autoantibody-producingability, which are derived from, preferably, a mammal such as a mouse,rat, guinea pig, hamster, rabbit, or human, or more preferably, a mouse,rat, or human.

For example, a mouse-derived myeloma P3/X63-AG8.653 (653),P3/NSI/1-Ag4-1 (NS-1), P3/X63-Ag8.U1 (P3U1), SP2/0-Ag14 (Sp2/0, Sp2),PAI, F0, NSO, or BW5147, rat-derived myeloma 210RCY3-Ag.2.3., orhuman-derived myeloma U-266AR1, GM1500-6TG-A1-2, UC729-6, CEM-AGR,D1R11, or CEM-T15 can be used as a myeloma for cell fusion.

Hybridomas producing monoclonal antibodies can be screened bycultivating hybridomas, for example, in microtiter plates and bymeasuring the reactivity of the culture supernatant in wells in whichhybridoma growth is observed, to the immunogen used for the immunizationmentioned above, for example, by an enzyme immunoassay such as RIA andELISA.

Monoclonal antibodies can be produced from hybridomas by cultivating thehybridomas in vitro or in vivo such as in the ascites fluid of a mouse,rat, guinea pig, hamster, or rabbit, preferably a mouse or rat, morepreferably mouse, and isolating the antibodies from the resultingculture supernatant or ascites fluid of a mammal.

Cultivating hybridomas in vitro can be performed depending on, e.g., theproperty of cells to be cultured, the object of the study, and thevarious conditions of the culture method, by using known nutrient mediaor any nutrient media derived from known basal media for growing,maintaining, and storing the hybridomas to produce monoclonal antibodiesin the culture supernatant.

Examples of basal media are low calcium concentration media such as Ham′F12 medium, MCDB153 medium, or low calcium concentration MEM medium, andhigh calcium concentration media such as MCDB104 medium, MEM medium,D-MEM medium, RPMI1640 medium, ASF104 medium, or RD medium. The basalmedia can contain, for example, sera, hormones, cytokines, and/orvarious inorganic or organic substances depending on the objective.

Monoclonal antibodies can be isolated and purified from the culturesupernatant or ascites fluid mentioned above by saturated ammoniumsulfate precipitation, euglobulin precipitation method, caproic acidmethod, caprylic acid method, ion exchange chromatography (DEAE orDE52), and affinity chromatography using an anti-immunoglobulin columnor a protein A column.

A “recombinant chimeric monoclonal antibody” is a monoclonal antibodyprepared by genetic engineering, and specifically means a chimericantibody such as a mouse/human chimeric monoclonal antibody whosevariable regions are derived from an immunoglobulin of a non-humanmammal (mouse, rat, hamster, etc.) and whose constant regions arederived from human immunoglobulin.

A constant region derived from human immunoglobulin has an amino acidsequence unique to each isotype such as IgG (IgG1, IgG2, IgG3, IgG4),IgM, IgA, IgD, and IgE. The constant region of the recombinant chimericmonoclonal antibody can be that of human immunoglobulin belonging to anyisotype. Preferably, it is a constant region of human IgG.

A chimeric monoclonal antibody can be produced, for example, as follows.Needless to say, the production method is not limited thereto.

A mouse/human chimeric monoclonal antibody can be prepared, referring toExperimental Medicine: SUPPLEMENT, Vol. 1.6, No. 10 (1988); and ExaminedPublished Japanese Patent Application No. (JP-B) Hei 3-73280. Namely, itcan be prepared by operably inserting the C_(H) gene (C gene encodingthe constant region of H chain) obtained from a DNA encoding humanimmunoglobulin downstream of active V_(H) genes (rearranged VDJ geneencoding the variable region of H chain) obtained from a DNA encoding amouse monoclonal antibody isolated from hybridoma producing the mousemonoclonal antibody, and the C_(L) gene (C gene encoding the constantregion of L chain) obtained from a DNA encoding human immunoglobulindownstream of active V_(L) genes (rearranged VJ gene encoding thevariable region of L chain) obtained from a DNA encoding a mousemonoclonal antibody isolated from hybridoma, into the same vector or adifferent vector in an expressible manner, followed by transforming hostcells with the expression vector, and then by cultivating thetransformants.

Specifically, DNAs are first extracted from mouse monoclonalantibody-producing hybridomas by the usual method, digested withappropriate restriction enzymes (for example, EcoRI and HindIII),electrophoresed (using, for example, 0.7% agarose gel), and analyzed bySouthern blotting. After an electrophoresed gel is stained, for examplewith ethidium bromide, and photographed, the gel is given markerpositions, washed twice with water, and soaked in 0.25 M HCl for 15minutes. Then, the gel is soaked in a 0.4 N NaOH solution for 10 minuteswith gentle stirring. The DNAs are transferred to a filter for 4 hoursby the usual method. The filter is recovered and washed twice with2×SSC. After the filter is sufficiently dried, it is baked at 75° C. for3 hours. After baking, the filter is treated with 0.1×SSC/0.1% SDS at65°C. for 30 minutes. Then, it is soaked in 3×SSC/0.1% SDS. The filterobtained is treated with a prehybridization solution in a plastic bag at65° C. for 3 to 4 hours.

Next, 32P-labeled probe DNA and a hybridization solution are added tothe bag and reacted at 65° C. about 12 hours. After hybridization, thefilter is washed under an appropriate salt concentration, reactiontemperature, and time (for example, 2×SSC/0.1% SDS, room temperature, 10minutes) . The filter is put into a plastic bag with a small volume of2×SSC and subjected to autoradiography after the bag is sealed.

Rearranged VDJ gene and VJ gene encoding H chain and L chain of a mousemonoclonal antibody are identified by Southern blotting mentioned above.The region comprising the identified DNA fragment is fractioned bysucrose density gradient centrifugation and inserted into a phage vector(for example, Charon 4A, Charon 28, λEMBL3, and λEMBL4). E. coli (forexample LE392 and NM539) is transformed with the phage vector togenerate a genomic library. The genomic library is screened by a plaquehybridization technique such as the Benton-Davis method (Science, Vol.196, pp. 180-182 (1977)) using appropriate probes (H chain J gene, Lchain (K) J gene, etc.) to obtain positive clones comprising rearrangedVDJ gene or VJ gene. By making a restriction map and determining thenucleotide sequence of the clones obtained, it is confirmed whethergenes comprising the desired, rearranged V_(H) (VDJ) gene or V_(L) (VJ)gene have been obtained.

Separately, human C_(H) gene and human C_(L) gene used for chimerizationare isolated. For example, when a chimeric antibody with human IgG1 isproduced, Cγ1 gene is isolated as a C_(H) gene, and Cκ gene as a C_(L)gene. These genes can be isolated from a human genomic library withmouse Cγ1 gene and mouse Cκ gene, corresponding to human Cγ1 gene andhuman Cκ gene, respectively, as probes, taking advantage of the highhomology between the nucleotide sequences of the mouse immunoglobulingene and the human immunoglobulin gene.

Specifically, DNA fragments comprising human Cκ gene and an enhancerregion are isolated from human λ Charon 4A HaeIII-AluI genomic library(Cell, Vol. 15, pp. 1157-1174 (1978)), for example, using a 3 kbHindIII-BamHI fragment of clone Ig146 (Proc. Natl. Acad. Sci. USA, Vol.75, pp. 4709-4713 (1978)) and a 6.8 kb EcoRI fragment of clone MEP10(Proc. Natl.Acad. Sci. USA, Vol. 78, pp. 474-478 (1981)) as probes. Inaddition, for example, after human fetal hepatocyte DNA is digested withHindIII and fractioned by agarose gel electrophoresis, a 5.9 kb fragmentis inserted into λ788 and then human Cγ1 gene is isolated with theprobes mentioned above.

Using mouse V_(H) gene, mouse V_(L) gene, human C_(H) gene, and humanC_(L) gene so obtained, and taking the promoter region and enhancerregion into consideration, human C_(H) gene is inserted downstream mouseV_(H) gene and human C_(L) gene is inserted downstream mouse V_(L) geneinto an expression vector such as pSV2gpt or pSV2neo with appropriaterestriction enzymes and DNA ligase by the usual method. In this case,chimeric genes of mouse V_(H) gene/human C_(H) gene and mouse V_(L)gene/human C_(L) gene can be respectively inserted into the sameexpression vector or into different expression vectors.

Chimeric gene-inserted expression vector(s) thus prepared are introducedinto myelomas that do not produce antibodies, for example, P3X63·Ag8·653cells or SP210 cells by the protoplast fusion method, DEAE-dextranmethod, calcium phosphate method, or electroporation method. Thetransformants are screened by cultivating in media containing a drugcorresponding to the drug resistance gene inserted into the expressionvector and, then, cells producing desired chimeric monoclonal antibodiesare obtained.

Desired chimeric monoclonal antibodies are obtained from the culturesupernatant of antibody-producing cells thus screened.

The “humanized monoclonal antibody (CDR-grafted antibody)” of thepresent invention is a monoclonal antibody prepared by geneticengineering and specifically means a humanized monoclonal antibodywherein a portion or the whole of the complementarity-determiningregions of the hypervariable region are derived from thecomplementarity-determining regions of the hypervariable region from amonoclonal antibody of an non-human mammal (mouse, rat, hamster, etc.),the framework regions of the variable region are derived from theframework regions of the variable region from human immunoglobulin, andthe constant region is derived from a constant region from human-derivedimmunoglobulin.

The complementarity-determining regions of the hypervariable regionexists in the hypervariable region in the variable region of an antibodyand means three regions which directly and complementary binds to anantigen (complementarity-determining residues, CDR1, CDR2, and CDR3).The framework regions of the variable region mean four comparativelyconserved regions lying upstream, downstream, or between the threecomplementarity-determining regions (framework region, FR1, FR2, FR3,and FR4).

In other words, a humanized monoclonal antibody means that in which allthe regions except a portion or the whole of thecomplementarity-determining regions of the hypervariable region of anon-human mammal-derived monoclonal antibody have been replaced withtheir corresponding regions derived from a human immunoglobulin.

The constant region derived from human immunoglobulin has an amino acidsequence unique to each isotype such as IgG (IgG1, IgG2, IgG3, IgG4),IgM, IgA, IgD, and IgE. The constant region of a humanized monoclonalantibody in the present invention can be that from human immunoglobulinbelonging to any isotype. Preferably, it is a constant region of humanIgG. The framework regions of the constant region derived from humanimmunoglobulin are not particularly limited.

A humanized monoclonal antibody can be produced, for example, asfollows. Needless to say, the production method is not limited thereto.

For example, a recombinant humanized monoclonal antibody derived frommouse monoclonal antibody can be prepared by genetic engineering,referring to Published Japanese Translation of International Publication(JP-WA) No. Hei 4-506458 and JP-A Sho 62-296890. Namely, at least onemouse H chain CDR gene and at least one mouse L chain CDR genecorresponding to the mouse H chain CDR gene are isolated from hybridomasproducing mouse monoclonal antibody, and human H chain gene encoding thewhole regions except human H chain CDR corresponding to mouse H chainCDR mentioned above and human L chain gene encoding the whole regionexcept human L chain CDR corresponding to mouse L chain CDR mentionedabove are isolated from human immunoglobulin genes.

The mouse H chain CDR gene(s) and the human H chain gene(s) so isolatedare operably inserted into an appropriate vector so that they can beexpressed. Similarly, the mouse L chain CDR gene(s) and the human Lchain gene(s) are operably inserted into another appropriate vector sothat they can be expressed. Alternatively, the mouse H chain CDRgene(s)/human H chain gene(s) and mouse L chain CDR gene(s)/human Lchain gene(s) can be operably inserted into the same expression vectorin an expressible manner. Host cells are transformed with the expressionvector thus prepared to obtain transformants producing humanizedmonoclonal antibody. By cultivating the transformants, a desiredhumanized monoclonal antibody is obtained from the culture supernatant.

The “human monoclonal antibody” is an immunoglobulin in which the entireregions comprising the variable and constant region of H chain, and thevariable and constant region of L chain constituting the immunoglobulinare derived from genes encoding human immunoglobulin.

The human antibody (preferably human monoclonal antibody) can beproduced by well known methods, for example, in the same way as theproduction method of polyclonal or monoclonal antibodies mentioned aboveby immunizing, with an antigen, a transgenic animal prepared byintegrating at least a human immunoglobulin gene into the gene locus ofa non-human mammal such as a mouse.

For example, a transgenic mouse producing human antibodies is preparedby the methods described in Nature Genetics, Vol. 7, pp. 13-21(1994);Nature Genetics, Vol. 15, pp. 146-156 (1997); JP-WA Hei 4-504365; JP-WAHei 7-509137; Nikkei Science, No. 6, pp. 40-50 (1995); WO94/25585;Nature, Vol. 368, pp. 856-859 (1994); and JP-WA No. Hei 6-500233.

In addition, a recently developed technique for producing ahuman-derived protein from the milk of a transgenic cow or pig can alsobe applied (Nikkei Science, pp. 78-84 (April, 1997)).

The expression “portion of an antibody” as used in the present inventionmeans a partial region of a monoclonal antibody as mentioned above. Itspecifically means F(ab′)₂, Fab′, Fab, Fv (variable fragment ofantibody), sFv, dsFv (disulfide stabilized Fv), or dAb (single domainantibody) (Exp. Opin. Ther. Patents, Vol. 6, No. 5, pp. 441-456 (1996)).

“F(ab′)₂” and “Fab′” can be produced by treating immunoglobulin(monoclonal antibody) with a protease such as pepsin and papain, andmeans an antibody fragment generated by digesting the immunoglobulinnear the disulfide bonds in the hinge regions existing between each ofthe two H chains. For example, papain cleaves IgG upstream of thedisulfide bonds in the hinge regions existing between each of the two Hchains to generate two homologous antibody fragments in which an L chaincomposed of V_(L) (L chain variable region) and C_(L) (L chain constantregion), and an H chain fragment composed of V_(H) (H chain variableregion) and C_(H)γ1 (γ1 region in the constant region of H chain) areconnected at their C terminal regions through a disulfide bond. Each ofsuch two homologous antibody fragments is called Fab′. Pepsin alsocleaves IgG downstream of the disulfide bonds in the hinge regionsexisting between each of the two H chains to generate an antibodyfragment slightly larger than the fragment in which the twoabove-mentioned Fab's are connected at the hinge region. This antibodyfragment is called F(ab′)₂. The expressions, “immunity of the intestinaltract”, “gastrointestinal immunity”, and “mucosal immunity” of thisinvention are used to express almost the same meaning.

A preferred example of a “disease accompanying abnormal immunity of theintestinal tract” of this invention may be an inflammatory bowel diseaseor an alimentary allergy.

A representative example of an “inflammatory bowel disease” of thisinvention is colitis (especially Ulcerative Colitis (UC)) or Crohn'sdisease (CD) , each having, for example, the following characteristics.

Inflammatory bowel diseases (IBD) can be classified into colitis(especially ulcerative colitis) and Crohn's disease. These diseasesfrequently develop in juveniles and are considered to be intractablechronic inflammatory diseases that repeat remission and recurrence and,having unknown causes.

Crohn's disease is a disease in which chronic granulomatousinflammations and ulcers occur in the entire digestive tract from theesophagus to the anus, mainly in the small intestine and largeintestine, showing symptoms such as abdominal pain, diarrhea, fever,abnormalities of the anus including hemorrhoids, and/or a decrease inbody weight. Histologically, a heavy infiltration of lymphocytes andnon-caseous epithelioid granuloma are observed, suggesting an abnormalreaction of T cells and antigen-presenting cells.

Colitis (especially ulcerative colitis) is a chronic inflammation thatdevelops locally in the large intestine. It mainly affects the mucousmembrane and forms sores and ulcerations. Histologically, a significantinfiltration of lymphocytes, plasma cells, macrophage, and mast cellsare observed in the mucous membrane and lamina propria mucosae, andcryptic ulcers accompanying an infiltration of neutrophils and adisappearance of goblet cells occur.

The existing pharmaceutical agents used for the treatment ofinflammatory bowel diseases of this invention refer to one or morearbitrary pharmaceutical agents clinically prescribed to treat colitis(ulcerative colitis and such) and Crohn's disease, and examples areadrenocortical hormones, and salazosulfapyridine.

The expression “pharmaceutically acceptable carrier” of this inventionincludes an excipient, a diluent, a filler, a decomposing agent, astabilizer, a preservative, a buffer, an emulsifier, an aromatic agent,a colorant, a sweetener, a viscosity-increasing agent, a flavor, asolubility-increasing agent, or some other additive. Using one or moreof such carriers, a pharmaceutical composition can be formulated intotablets, pills, powders, granules, injections, solutions, capsules,troches, elixirs, suspensions, emulsions, syrups, etc.

The pharmaceutical composition can be administered orally orparenterally. Other forms for parenteral administration include asolution for external application, suppository for rectaladministration, and pessary, prescribed by the usual method, whichcomprises one or more active ingredients.

The dosage can vary depending on the age, sex, weight, and symptoms of apatient, effect of treatment, administration route, period of treatment,the kind of active ingredient (the “substance” according to the presentinvention, mentioned above) contained in the pharmaceutical composition,etc. Usually, the pharmaceutical composition can be administered to anadult in a dose of 10 μg to 1000 mg (or 10 μg to 500 mg) per oneadministration. Depending on various conditions, a dosage less than thatmentioned above may be sufficient in some cases and a dosage more thanthat mentioned above may be necessary in others.

In the case of an injection, it can be produced by dissolving orsuspending an antibody in a non-toxic, pharmaceutically acceptablecarrier such as physiological saline or commercially available distilledwater for injection adjusting the concentration in the range of 0.1 μgantibody/ml carrier to 10 mg antibody/ml carrier. The injection thusproduced can be administered to a human patient in need of treatment inthe dose range of 1 μg to 100 mg/kg body weight, preferably in the rangeof 50 μg to 50 mg/kg body weight, one or more times a day. Examples ofadministration routes are medically appropriate administration routessuch as intravenous injection, subcutaneous injection, intradermalinjection, intramuscular injection, intraperitoneal injection, or such,preferably intravenous injection.

The injection can also be prepared into a non-aqueous diluent (forexample, propylene glycol, polyethylene glycol, vegetable oil such asolive oil, and alcohol such as ethanol), suspension, or emulsion.

The injection can be sterilized by filtration with a bacteria-filteringfilter, by mixing a bacteriocide, or by irradiation. The injection canbe produced in such a manner that it is prepared at the time of use.Namely, it is freeze-dried to be a sterile solid composition that can bedissolved in sterile distilled water for injection or another solventbefore use.

Using the pharmaceutical compositions of this invention, diseases thatmay be caused by abnormal immunity of the intestinal tract, morespecifically, inflammatory bowel diseases (especially Crohn's diseaseand colitis (especially ulcerative colitis)) and alimentary allergiescan be suppressed, prevented, and/or treated.

Furthermore, by using the pharmaceutical composition of this invention,it is possible to enhance the therapeutic effect of existingpharmaceutical agents that are prescribed for the treatment of suchinflammatory diseases.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the suppressive effect of an anti-AILIM antibody on theonset of an inflammatory bowel disease when the antibody is continuouslyadministered (before the onset or after disease progression), and thetherapeutic effect on the inflammatory bowel disease by theadministration of the anti-AILIM antibody, determined using weight loss,which is a characteristic of colitis, as an index.

FIG. 2 is a photograph showing the state of the large intestine ofnormal mice that have not developed the inflammatory bowel disease, thatof mice affected by the inflammatory bowel disease, and that of mice inwhich the onset of the inflammatory bowel disease is found to besuppressed by the administration of the anti-AILIM antibody,respectively.

FIG. 3 shows the suppressive effect of the anti-AILIM antibody on theonset of the inflammatory bowel disease when the antibody iscontinuously administered, as determined using weight loss, which is acharacteristic of colitis, as an index. : negative control antibody(n=20) ◯: anti-AILIM/ICOS antibody (n=20) □: anti-B7RP-1 antibody (n=7)▪: administration of negative control antibody to a BALB/c scid/scidmouse to which CD4⁺CD45RB^(low) T cells have been introduced instead ofCD4⁺CD45RB^(high) T cells (n=7)

FIG. 4 shows the degree of severity of colitis expressed as histologicalscores.

FIG. 5 shows the number of CD4⁺ cells that infiltrated into the colonicmucosal layer (lamina propria).

FIG. 6 shows the degree of apoptosis of cells in colon tissues.

FIG. 7 shows the therapeutic effect of the administration of theanti-AILIM antibody (administration after disease progression) on theinflammatory bowel disease, as determined using weight loss, which is acharacteristic of colitis, as an index : negative control antibody □:anti-AILIM/ICOS antibody

FIG. 8 shows the degree of severity of colitis expressed as histologicalscores.

FIG. 9 shows the number of CD4⁺ cells that infiltrated into the colonicmucosal layer (lamina propria).

FIG. 10 shows the suppressive effect of the continuously administeredanti-AILIM antibody on the onset of the inflammatory bowel disease, asdetermined using weight loss, which is a characteristic of colitis, asan index. : negative control antibody (n=7) □: anti-AILIM/ICOS antibody(n=7)

FIG. 11 shows the degree of severity of colitis expressed ashistological scores.

FIG. 12 shows the number of CD4⁺ cells that infiltrated into the colonicmucosal layer (lamina propria).

BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the present invention is specifically illustrated withreference to Examples, but it is not to be construed as being limitedthereto.

EXAMPLE 1 The Therapeutic Effect on Colitis in a Mouse Colitis Model<Trial 1> <1-1>Animals

BALB/c scid/scid mice, severely immunodeficient mice (6-8 weeks oldfemales; CLEA Japan), and normal BALB/c mice (6-8 weeks old males; CLEAJapan) were used.

<1-2>Preparation of an Anti-mouse AILIM Monoclonal Antibody

The preparation was done as follows.

Using the cDNA encoding the full length amino acid sequence of thepreviously reported mouse AILIM (Int. Immunol., Vol. 12, No. 1, p.51-55, 2000), a transformed cell expressing mouse AILIM was preparedaccording to standard methods using genetic recombination technology.

The transformed cell was homogenized and ultra-centrifuged (100,000× g),and the centrifuged residue containing the cell membrane fraction wascollected and suspended in PBS. The obtained cell membrane fraction wasinjected together with complete Freund's adjuvant into the foot pad of aWistar rat for the initial immunization (day 0). In addition, the cellmembrane fraction was administered as an antigen into the foot pad withintervals, on day 7, day 14, and day 28. Two days after the finalimmunization, lymph node cells were collected.

The lymph node cells and mouse myeloma cells PAI (JCR No. B 0113; Res.Disclosure, Vol. 217, p. 155, 1982) were mixed in a 5:1 ratio, and amonoclonal antibody-producing hybridoma was prepared by fusing the cellsusing polyethylene glycol 4000 (Boehringer Mannheim) as the fusingagent. Hybridoma selection was performed by culturing in aHAT-containing ASF104 medium (Ajinomoto) containing 10% fetal bovineserum and aminopterin.

The fluorescence intensities of cells stained by reacting the culturesupernatants of each hybridoma with the above-mentioned recombinantmouse AILIM-expressing transfected cells and then reacting them withFITC-labeled anti-rat IgG (Cappel) were measured using the EPICS-ELITEflow cytometer to confirm the reactivity of the monoclonal antibodiesproduced in the culture supernatant of each hybridoma against mouseAILIM. As a result, several hybridomas that produced monoclonalantibodies having reactivity towards mouse AILIM were obtained.

One of these hybridomas was named “B10.5”. This hybridoma (10⁶ to 10⁷cells/0.5 mL/mouse each) was injected intraperitoneally to an ICR nu/numouse (female, 7 to 8 weeks old). Ten to twenty days later, laparotomywas performed on the mouse under anesthesia, and a large quantity of ratanti-mouse AILIM monoclonal antibody (IgG2a) was obtained from theascites according to standard procedures. Hereinafter, this antibody issimply referred to as “anti-AILIM antibody”.

<1-3> Induction of the Inflammatory Bowel Disease

As described below, an inflammatory bowel disease (colitis) was inducedby introducing CD45RB^(high) into a BALB/c scid/scid mouse according toa previously reported method. Accompanying the onset and progression ofthe inflammatory bowel disease, a significant weight loss is known tooccur 3 to 5 weeks after the introduction of CD45RB^(high) T cells inthis inflammatory bowel disease model.

CD4⁺ T cells were separated and obtained from mononuclear cells derivedfrom spleens of healthy BALB/c mice by using a MACS magnetic separationsystem (Miltenyi Biotec) with an anti-CD4 antibody (L3T4). Morespecifically, spleen cells isolated from the mice were cultured at 4° C.for 30 min with magnetic beads to which an anti-CD4 antibody is bound,and then, these cells were washed and enriched by passing through amagnetic flow column.

After labeling the obtained CD4⁺ T cells (purity was confirmed to be96-97% using a flow cytometer) with an anti-mouse CD4 antibody (RM4-5;PharMingen) labeled with phycoerythrin (PE), and an anti-CD4 antibody(16A; PharMingen) labeled with fluorescein isothiocyanate (FITC), theywere sorted using a FACS Vantage (Becton Dickinson), and fractioned intoT cells having a high expression of CD45RB (CD45RB^(high)) and T cellshaving a low expression of CD45RB (CD45RB^(low) ).

Next, in order to induce colitis in the BALB/c scid/scid mice, theobtained CD45RB^(high) T cells (5×10⁵ cells/200 μL PBS) wereadministered intraperitoneally (i.p.) to the mice.

<1-4> Administration of the Anti-AILIM Antibody

Each group of the above-mentioned SCID mice to which CD45RB^(high) Tcells had been introduced was treated as follows.

Group 1

The anti-AILIM antibody (250 μg/250 μL PBS) was administeredintraperitoneally immediately after the introduction of CD45RB^(high) Tcells (first administration), and thereafter continuously every week ata frequency of 3 times/week.

Group 2

The negative control antibody (rat IgG, Sigma, 250 μg/250 μl PBS) wasadministered intraperitoneally immediately after the introduction ofCD45RB^(high) T cells (first administration), and thereaftercontinuously every week at a frequency of 3 times/week. Simultaneously,after the 8th week from immediately after the introduction ofCD45RB^(high) T cells (first administration) and onwards, in addition tothe negative control antibody, the anti-AILIM antibody (250 μg/250 μLPBS) was intraperitoneally administered continuously every week at asimilar frequency.

Group 3

The negative control antibody (rat IgG, Sigma, 250 μg/250 μl PBS) wasintraperitoneally administered from immediately after the introductionof CD45RB^(high) T cells (first administration) and thereaftercontinuously every week at a frequency of 3 times/week.

The degree of progression of the inflammatory bowel disease, and thedegree of the suppression and treatment of the onset and progression ofthe disease due to the anti-AILIM antibody were analyzed by measuringthe body weight of each group over time from immediately before theintroduction of T cells.

The results are shown in FIG. 1.

Therefore, as expected, a significant weight loss accompanying theprogression of the inflammatory bowel disease occurred in the group towhich only the negative control antibody was administered (Group 3).However, absolutely no weight loss was observed and the onset ofinflammatory bowel disease was completely suppressed in the group towhich the anti-AILIM antibody was continuously administered fromimmediately after the introduction of CD45RB^(high) T cells (Group 1).

Furthermore, in the group to which the negative control antibody alonewas administered from immediately after the introduction ofCD45RB^(high) T cells to week 7, and from week 8 the anti-AILIM antibodywas administered in addition to the negative control antibody (Group 2),a significant increase (recovery) in body weight was observed fromimmediately after the initiation of anti-AILIM antibody administration,compared to the group to which only the negative control antibody alonewas administered even after week 8 and onwards (Group 3). Therefore, theanti-AILIM antibody was found to cure inflammatory bowel disease.

Furthermore, the degree of progression of the inflammatory boweldisease, as well as the degree of suppression and treatment of the onsetand progression of the disease by the anti-AILIM antibody were analyzedby collecting the large intestines 6 weeks after T cell introductionfrom some mice of Group 1 and Group 3, and examining their state withthe naked eye. As a normal control, a similar observation was carriedout on the large intestine collected from BALB/c scid/scid mouse towhich no CD45RB^(high) T cells were introduced and no antibodies wereadministered.

The results are shown in FIG. 2.

As a result, involution of the large intestine (thickening andshortening of the intestinal tract) accompanying progression ofinflammatory bowel disease, and untreated stools were observed in thegroup to which the negative control antibody was administered (Group 3).However, the state of the large intestine of the group to whichanti-AILIM antibody was administered (Group 1) was similar to that ofthe large intestine of the normal control, and the anti-AILIM antibodywas revealed to significantly suppress the onset and progression ofinflammatory bowel disease.

Example 2 Therapeutic Effect on Inflammatory Bowel Disease in the MouseColitis Model <Trial 2> <2-1>Animals

Immunodeficient BALB/c scid/scid mice , C57BL/6 scid/scid mice, andnormal BALB/c mice (all male, 6 to 8 weeks old, CLEA Japan) were used.

<2-2>Monoclonal Antibodies

A monoclonal antibody against mouse AILIM (ICOS), a monoclonal antibodyagainst mouse B7RP-1 which is a ligand of mouse AILIM (ICOS), and anegative control antibody were used.

B10.5 produced as described above was used for the anti-mouse AILIM/ICOSmonoclonal antibody.

The anti-mouse B7RP-1 monoclonal antibody was produced in the followingmanner. An SD rat was immunized with recombinant L cells that expressmouse B7RP-1 produced according to standard procedures. Spleen cells ofthe immunized rat were obtained, and by cell fusion with myeloma cellsaccording to standard procedures, hybridomas were produced. Using theculture supernatant of each of the hybridomas, the degree of the bindingof anti-mouse B7RP-1 monoclonal antibody contained in the culturesupernatant to recombinant cells (NRK cells) that express mouse B7RP-1was measured by EIA, and hybridomas producing an antibody that bind tomouse B7RP-1 were selected. Anti-mouse B7RP-1 monoclonal antibody usedfor the examination was prepared from the culture supernatant of theselected hybridomas. Furthermore, it was confirmed that the anti-mouseB7RP-1 monoclonal antibody has an activity to inhibit the binding ofmouse AILIM-Ig (fusion polypeptide comprising the soluble region ofmouse AILIM/ICOS and Fc of an immunoglobulin) to recombinant cells thatexpress mouse B7RP-1.

Rat IgG (Sigma) that does not react with mouse AILIM/ICOS or B7RP-1 wasused as the negative control antibody.

<2-3> Induction of colitis (inflammatory bowel disease)

The inflammatory bowel disease colitis was induced by introducing CD4⁺CD45RB^(high) T cells into BALB/c scid/scid mice according to anexisting report (J. Immunol., Vol. 164, p. 4878-4882, 2000).

CD4⁺ T cells were separated from the spleen cells of normal BALB/c miceusing the anti-CD4 (L3T4) MACS magnetic separation system (MiltenyiBiotec) following the attached instructions. The CD4⁺ T cells (purity of96-97%, analyzed by FACS) were labeled with a PE-labeled anti-mouse CD4antibody (RM4-5; PharMingen) and a FITC-labeled anti-CD45RB antibody(16A; PharMingen), and were sorted into CD45RB^(high) T cells andCD45RB^(low) T cells using FACS Vantage (Becton Dickinson).

The inflammatory bowel disease (colitis) was induced by intraperitonealadministration of CD45RB^(high) T cells (5×10⁵ cells/200 μL PBS) intoBALB/c scid/scid mice.

<2-4>Treatment of Inflammatory Colitis by the Anti-AILIM Antibody

The Anti-mouse AILIM/ICOS monoclonal antibody (250 μg/250 μL PBS), theanti-mouse B7RP-1 monoclonal antibody (250 μg/250 μL PBS), or thecontrol antibody (250 μg/250 μL PBS) was administered intraperitoneallyat a frequency of 3 times per week for 7 weeks from the time ofintroduction (week 0) of CD45RB^(high) T cells to mice produced asdescribed above in which inflammatory colitis was induced.

On the other hand, to investigate the therapeutic effect of theanti-AILIM/ICOS antibody under the state of progressed inflammatorycolitis, the anti-AILIM/ICOS monoclonal antibody was administered (at aconcentration of 250 μg/body; 3 times/week; i.p.) under conditionssimilar to that mentioned above from 3 weeks after the introduction ofCD45RB^(high) T cells to mice produced as described above in whichinflammatory colitis had been induced. Seven weeks after theintroduction of CD45RB^(high) T cells, the test animals were sacrificedand the state of inflammation in the large intestine was analyzed.

<2-5>Introduction of Pathogenic CD4³⁰ T Cells from Mice with Colitis toMice

From the lamina propria (LP) of the large intestine of BALB/c scid/scidmice to which CD4⁺ CD45RB^(high) T cells had been introduced (adoptivetransfer), CD4⁺ cells (LP CD4⁺ T cells) were isolated using theabove-mentioned MACS magnetic beads 7 weeks after the introduction ofthe T cells. The purity of the isolated LP CD4⁺ T cells was confirmed tobe 95% or more by FACS.

The LP CD4⁺ T cells (1×10⁶ cells/200 μL PBS; i.p.) were administered toBALB/c scid/scid mice. Next, the anti-AILIM/ICOS antibody (250 μg/250 μLPBS) or the negative control antibody (rat IgG; 250 μg/250 μL PBS) wasadministered at a frequency of 3 times per week to the test mice. Fourweeks after the administration of LP CD4⁺ T cells, the mice wereeuthanized, the large intestines were removed and histologicallyanalyzed.

<2-6>Histological Examination and Immunohistochemical Staining

Large intestine tissue samples collected from each of the aforementionedtest animals were immobilized in PBS containing 6% formalin.Paraffin-immobilized large intestine tissue sections (5 μm) were stainedby hematoxylin and eosin (HE staining). Each of the produced tissuesection specimens was analyzed. The degree of inflammation of the largeintestine was converted to scores according to an existing report(Gastroenterology, Vol. 119, p. 715-723, 2000).

On the other hand, the large intestine samples for immunohistochemicalstaining analysis were immobilized in OCT compound, frozen in liquidnitrogen, and stored at −80° C. Staining of the tissue sections wasperformed by the avidin-biotin complex method.

The tissue sections (6 μm) were incubated with biotinylated anti-mouseAILIM/ICOS monoclonal antibody, biotinylated anti-mouse B7RP-1monoclonal antibody, biotinylated anti-mouse CD4 monoclonal antibody(RM4-5; rat IgG1; PharMingen) , biotinylated anti-mouse F4/80 monoclonalantibody (rat IgG2; PharMingen), or biotinylated isotype-matched controlantibody (PharMingen). The biotinylated antibody was detected withstreptavidin-biotinylated horseradish peroxidase complex (Vectastain ABCKit; Vector), and was visualized with diaminobenzidine. Next, each ofthe sections was counter stained with hematoxylin.

<2-7>Detection of Apoptotic Cells

Apoptotic cells in frozen tissue sections were detected using the ApoTagKit (Intergen) according to the previously reported TUNEL method. Byobserving under a microscope, TUNEL positive cells in the tissuesections were quantified by calculating the TUNEL positive cells in 500lamina propria mononuclear cells (LPMC) that had infiltrated into 5parts of one section. The percentage of TUNEL positive cells in 500 LPMCwas taken to be the apoptosis index.

<2-8>Results

The results are shown in FIG. 3 to FIG. 12.

<2-8-1>Suppression of Colitis by Administration of Anti-AILIM/ICOSAntibody

In the test mice (control mice) to which the negative control antibody(rat IgG) had been administered, severe colitis developed 4 to 7 weeksafter the introduction of CD45RB^(high) T cells, and not only asignificant weight loss (FIG. 3), but also a significant thickening ofthe intestinal wall of the large intestine accompanied by diarrhea andinflammation was observed.

In the control mice, the average value (on week 7) of histologicalscores (indicating the severity of colitis) characterized by transmuralinflammation in which a large number of lymphocytes are observed in thelamina propria and submucosa, and prominent epithelial hyperplasiaaccompanying a decrease of goblet cells was 5.9±1.2 (FIG. 4).

On the other hand, mice treated with the anti-AILIM/ICOS antibody wereabsolutely healthy, and signs of colitis and thickening of the colonicwall was not observed, and weight gain was observed over time(FIG. 3).Furthermore, the histological score of the colonic wall tissue was0.8±0.7 in these anti-AILIM/ICOS antibody-treated mice (n=7), and clearmorbid changes could not be observed (FIG. 4).

The average number of CD4⁺ T cells observed in the lamina propria of thelarge intestine of mice with colitis (control mice) that have developedan inflammation was 44±9×10⁵ cells/large intestine, whereas in micetreated with the anti-AILIM/ICOS antibody, it was 21±3×10⁵ cells/largeintestine (p<0.01) (FIG. 5).

<2-8-2>Induction of Apoptosis in Tissue-infiltrating Mononuclear Cellsby the Anti-AILIM/ICOS Antibody

In the large intestine of mice to which the anti-AILIM/ICOS antibody wasadministered, a significant increase of apoptotic cells (most of themwere tissue-infiltrating mononuclear cells) was observed compared tothat in mice treated with negative control antibody (control mice).

The apoptosis index as shown by quantitative analysis of the apoptoticcells in the large intestine was significantly high in mice to which theanti-AILIM/ICOS antibody was administered (n=5) compared to that in miceto which control antibody was administered (control mouse) (FIG. 6).

These results showed that the suppressive effect on colitis due to thetreatment with anti-AILIM/ICOS antibody is attributed to the depletionof pathogenic T cells that express AILIM (ICOS).

<2-8-3>Suppressive Effect on Colitis due to Administration ofAnti-AILIM/ICOS Antibody after Progression of Symptoms

Weight loss and infiltration of lymphocytes to the colonic tissues, i.e.wasting disease which is one of the symptoms in the above-mentionedcolitis model mouse, start 3 weeks or so and 2 weeks or so after theintroduction of CD₄₅RB^(high) T cells, respectively. Therefore, asmentioned above, the administration of the anti-AILIM/ICOS antibody wasinitiated 3 weeks after the introduction of the T cells.

In the anti-AILIM/ICOS antibody-administered group, a significantimprovement in weight loss was observed (FIG. 7) and diarrhea was notseen.

Histological analysis of the colonic tissue sections of mice from eachgroup showed that granulomatous inflammation, lymphocytic infiltration,and thickening of the epithelium were significantly reduced in thecolonic tissues of the group to which the anti-AILIM/ICOS antibody wasadministered, compared to that of the mice to which the negative controlantibody was administered (control mice) . Changes in inflammation wereobserved in the lamina propria, and in certain cases, in lesionsaccompanying a mild infiltration of lymphocytes into the submucosa, butnot in the muscular layer. Furthermore, the above-mentioned histologicalscore indicating the severity of colitis (the large intestines werecollected on week 7) was significantly decreased in the group ofanti-AILIM/ICOS antibody-administered mice (1.62±0.81) compared to thatof the group of negative control antibody-administered mice (controlmice; 5.93±1.23) (p<0.05; FIG. 8). Furthermore, the number ofinfiltrating CD4⁺ T cells (week 7) was significantly decreased in theanti-AILIM/ICOS antibody-administered mice (5.50±0.7×10⁶ cells) comparedto that of the control mice (31.3±7.7×10⁶ cells) (p<0.05; FIG. 9).

<2-8-4>Suppressive Effect of the Anti-AILIM/ICOS Antibody on ColitisInduced by the Introduction of Lamina Propria CD4⁺ T Cells Derived froma Colitis Donor

To investigate the therapeutic effect of anti-AILIM/ICOS antibody onpathogenic T cells, as described above, colitis was induced byadministering LP CD4³⁰ T cells derived from mice with colitis to BALB/cscid/scid mice, followed by the administration of the anti-AILIM/ICOSantibody.

In the anti-AILIM/ICOS antibody-administered mice, weight loss (FIG. 10)was significantly improved. In addition, the histological scoreindicating the severity of colitis (the large intestines were collectedon week 4; FIG. 11) and infiltration of CD4⁺ cells into the coloniclamina propria (week 4) (FIG. 12) were also significantly decreasedcompared to that of the negative control antibody-administered mice(control mice).

INDUSTRIAL APPLICABILITY

The pharmaceutical compositions of the present invention (particularlypreferably comprising a substance having an activity to induce celldeath, apoptosis, or depletion of cells that express AILIM/ICOS) areextremely useful for suppressing, preventing, and/or treating diseasesthat may be caused by an abnormal immunity of the intestinal tract, morespecifically, inflammatory bowel diseases (especially Crohn's diseaseand colitis (ulcerative colitis and such)) and alimentary allergies.

The pharmaceutical compositions of this invention can enhance thetherapeutic effect on inflammatory bowel diseases and alimentaryallergies when used in combination with existing pharmaceutical agentsthat are prescribed for the treatment of such inflammatory diseases andalimentary allergies.

Furthermore, a pharmaceutical composition comprising a human antibodyagainst AILIM, which is included as a part of the pharmaceuticalcomposition of this invention, is extremely useful as a drug since itdoes not cause any side effects such as allergies as seen whenadministering antibodies derived from mice to humans.

1-10. (canceled)
 11. A method of suppressing or treating a disease thataccompanies abnormal immunity of the intestinal tract in a subject, themethod comprising administering to the subject an effective amount of acomposition comprising (a) an antibody or a portion thereof that bindsto human AILIM, and (b) a pharmaceutically acceptable carrier.
 12. Themethod of claim 11, wherein the disease is an alimentary allergy.
 13. Amethod of suppressing or treating an inflammatory bowel disease in asubject, the method comprising administering to the subject an effectiveamount of a composition comprising (a) an antibody or a portion thereofthat binds to human AILIM, and (b) a pharmaceutically acceptablecarrier.
 14. The method of claim 13, wherein the inflammatory boweldisease is colitis.
 15. The method of claim 13, wherein the inflammatorybowel disease is Crohn's disease.
 16. The method of claim 11, whereinthe antibody or portion thereof is a monoclonal antibody.
 17. The methodof claim 16, wherein the monoclonal antibody is a chimeric monoclonalantibody, a humanized monoclonal antibody, or a human monoclonalantibody.
 18. The method of claim 11, wherein the antibody or portionthereof is an F(ab′)₂ fragment, an Fab′ fragment, an Fab fragment, an Fvfragment, or a single domain antibody.
 19. The method of claim 12,wherein the antibody or portion thereof is a monoclonal antibody. 20.The method of claim 19, wherein the monoclonal antibody is a chimericmonoclonal antibody, a humanized monoclonal antibody, or a humanmonoclonal antibody.
 21. The method of claim 12, wherein the antibody orportion thereof is an F(ab′)₂ fragment, an Fab′ fragment, an Fabfragment, an Fv fragment, or a single domain antibody.
 22. The method ofclaim 13, wherein the antibody or portion thereof is a monoclonalantibody.
 23. The method of claim 22, wherein the monoclonal antibody isa chimeric monoclonal antibody, a humanized monoclonal antibody, or ahuman monoclonal antibody.
 24. The method of claim 13, wherein theantibody or portion thereof is an F(ab′)₂ fragment, an Fab′ fragment, anFab fragment, an Fv fragment, or a single domain antibody.
 25. Themethod of claim 14, wherein the antibody or portion thereof is amonoclonal antibody.
 26. The method of claim 25, wherein the monoclonalantibody is a chimeric monoclonal antibody, a humanized monoclonalantibody, or a human monoclonal antibody.
 27. The method of claim 14,wherein the antibody or portion thereof is an F(ab′)₂ fragment, an Fab′fragment, an Fab fragment, an Fv fragment, or a single domain antibody.28. The method of claim 15, wherein the antibody or portion thereof is amonoclonal antibody.
 29. The method of claim 28, wherein the monoclonalantibody is a chimeric monoclonal antibody, a humanized monoclonalantibody, or a human monoclonal antibody.
 30. The method of claim 15,wherein the antibody or portion thereof is an F(ab′)₂ fragment, an Fab′fragment, an Fab fragment, an Fv fragment, or a single domain antibody.